Abstract Reactive electrophile species constantly modify biomolecules and impact human health in ways that are still poorly understood. This research program establishes the methodological groundwork to rigorously test hypotheses regarding reactive electrophilic species and to develop new covalent irreversible drugs. Bioactive electrophiles such as ?,?-unsaturated carbonyls are present in the environment and diet, but they are also produced endogenously during metabolism and oxidative stress. They have been linked to the etiology of diverse pathological states such as atherosclerosis, cancer, neurodegeneration, and inflammation. Understanding the roles of reactive electrophilic species in homeostasis and disease will provide important guidance for disease prevention and the development of new therapeutics. However, a lack of research tools has impeded such endeavors and many studies in this field lack experimental rigor. The problem is that these reactive electrophilic species irreversibly modify a large number of biomolecules with very little control by the scientist. The program will close critical methodological gaps in the research on the effects of electrophiles in biology. Anticipated deliverables are methods that enable the controlled generation of such electrophiles at specific times and locations. Furthermore, methods to controllably reverse the formed covalent adducts of such electrophiles will be developed. Additionally, screening technologies to develop molecules that target specific protein residues will be established. The methods are innovative because they allow answering important biomedical questions that are currently out of reach. The program?s preliminary studies have established a solid foundation to achieve these goals. We have developed chemical reactions that allow generating ?,?-unsaturated carbonyls on-demand in living systems, and we have established DNA-encoded libraries for the discovery of bioactive species. With these unique capabilities in hand, the research program is in the position to advance the understanding of reactive electrophilic species in biology and their impact on disease. To demonstrate their utility, we will apply them to study the involvement of 4-hydroxynonenal in atherosclerosis and study the dynamics of adaptive states associated with resistance to KRASG12C inhibitors of pancreatic cancer. We will take steps to ensure that the developed methods will be available to a broad range of scientists to maximize the impact of the program.